Wolman disease and cholesteryl ester storage disease are characterized by a deficiency in activity of lysomal acid lipase which results in massive accumulation of cholesteryl esters and triglycerides in most tissues of the body. Cholesteryl esters and triglycerides are derived from plasma lipoproteins taken up by the cells and are substrates for acid lipase. Acid lipase is responsible for the hydrolysis of cholesteryl esters and triglycerides in the lysosomes.
If plasma cholesterol levels are lowered sufficiently, then cholesteryl ester and triglyceride accumulation in the lysosomes and the consequences of the accumulation could be minimized.
Wolman disease is the more severe of the two diseases and is almost always fatal before the age of 1 year. In contrast, cholesteryl ester storage disease may go undetected until adulthood by which time lipid deposition is widespread. Hyperbetalipoproteinemia is common in cholesteryl ester storage disease, and premature atherosclerosis may be severe.
To date, there has been no specific therapy for acid lipase deficiency other than attempts at suppression of cholesterol synthesis and apolipoprotein B production by 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors in combination with cholestyramine treatment and a diet excluding foods rich in cholesterol and triglycerides. The above apparently provided improvement in only one or two cases of cholesteryl ester storage disease. Thus, for the most part, Wolman disease and cholesteryl ester storage disease have been untreatable.
See Scriver, C. R. et al "The Metabolic and Molecular Bases of Inherited Disease", Vol. II (1995), Chap. 82, "Acid Lipase Deficiency: Wolman Disease and Cholesteryl Ester Storage Disease", pp. 2563-2587.
Until now, there have not been any therapeutic agents available which could lower plasma cholesterol levels sufficiently to minimize cholesteryl ester and triglyceride accumulation in the lysosomes.
The microsomal triglyceride transfer protein (MTP) catalyzes the transport of triglyceride (TG), cholesteryl ester (CE), and phosphatidylcholine (PC) between small unilamellar vesicles (SUV). Wetterau & Zilversmit, Chem. Phys. Lipids 38, 205-22 (1985). When transfer rates are expressed as the percent of the donor lipid transferred per time, MTP expresses a distinct preference for neutral lipid transport (TG and CE), relative to phospholipid transport. The microsomal triglyceride transfer protein from bovine liver has been isolated and extensively characterized (1). This has led to the cloning of cDNA expressing the protein from several species, including humans (2). MTP is composed of two subunits. The small subunit is the previously characterized multifunctional protein, protein disulfide isomerase. This is supported by biochemical analysis of the protein (3) as well as co-expression studies performed in insect Sf9 cells using the baculovirus expression system. Expression of soluble active MTP requires the co-expression of PDI and the unique large subunit of MTP (4).
1: Wetterau, J. R. and Zilversmit, D. B. (1985) Chem. Phys. Lipids 38, 205-222. PA0 Wetterau, J. R., et al, (1990) J. Biol. Chem. 265, 9800-9807. PA0 Wetterau, J. R., et al, (1991) Biochemistry 30, 4406-4412. PA0 Atzel, A., and Wetterau, J. R. (1993) Biochemistry 32, 10444-10450. PA0 Atzel, A., and Wetterau, J. R. (1994) Biochemistry 33, 15382-15388. PA0 Jamil, H., et al, (1995) J. Biol. Chem. 270, 6549-6554. PA0 2. Sharp, D. et al, (1993) Nature 365, 65-69. PA0 Lin, M. C. M., et al, J. Biol. Chem. 269, 29138-29145. PA0 Nakamuta, M., et al, (1996) Genomics 33, 313-316. PA0 3. Wetterau, J. R., et al, (1990) J. Biol. Chem. 265, 9800-9807. PA0 Wetterau, J. R., et al, (1991) Biochemistry 30, 9728-9735. PA0 4. Ricci, B., et al, (1995) J. Biol. Chem. 270, 14281-14285. PA0 5. Wetterau, J. R., et al, (1992) Science 258, 999-1001. PA0 Sharp, D., et al, (1993) Nature 365, 65-69. PA0 Ricci, B., et al, (1995) J. Biol. Chem. 270, 14281-14285. PA0 Shoulders, C. C., et al, (1993) Hum. Mol. Genetics 2, 2109-2116. PA0 Narcisi, T. M. E., et al, (1995) Am. J. Hum. Genet. 57, 1298-1310. PA0 Rehberg, E. F., et al, J. Biol. Chem (in press).
In vitro, MTP catalyzes the transport of lipid molecules between phospholipid membranes. Presumably, it plays a similar role in vivo, and thus plays some role in lipid metabolism. The subcellular (lumen of the microsomal fraction) and tissue distribution (liver and intestine) of MTP have led to speculation that it plays a role in the assembly of plasma lipoproteins, as these are the sites of plasma lipoprotein assembly. Wetterau & Zilversmit, Biochem. Biophys. Acta 875, 610-7 (1986). The ability of MTP to catalyze the transport of TG between membranes is consistent with this hypothesis, and suggests that MTP may catalyze the transport of TG from its site of synthesis in the endoplasmic reticulum (ER) membrane to nascent lipoprotein particles within the lumen of the ER.
Abetalipoproteinemia is an autosomal recessive disease characterized by a virtual absence of plasma lipoproteins which contain apolipoprotein B (apoB). Kane & Havel in The Metabolic Basis of Inherited Disease, Sixth edition, 1139-64 (1989). Plasma TG levels may be as low as a few mg/dL, and they fail to rise after fat ingestion. Plasma cholesterol levels are often only 20-45 mg/dL. These abnormalities are the result of a genetic defect in the assembly and/or secretion of very low density lipoproteins (VLDL) in the liver and chylomicrons in the intestine. The molecular basis for this defect had not been previously determined. In subjects examined, triglyceride, phospholipid, and cholesterol synthesis appear normal. At autopsy, subjects are free of atherosclerosis. Schaefer et al., Clin. Chem. 34, B9-12 (1988). A link between the apoB gene and abetalipoproteinemia has been excluded in several families. Talmud et al., J. Clin. Invest. 82, 1803-6 (1988) and Huang et al., Am. J. Hum. Genet. 46, 1141-8 (1990).
Recent reports (5) demonstrate that the defect causing abetalipoproteinemia is in the MTP gene, and as a result, the MTP protein. When examined, individuals with abetalipoproteinemia have no MTP activity, as a result of mutations in the MTP gene, some of which have been characterized. These results indicate that MTP is required for the synthesis of apoB containing lipoproteins, such as VLDL, the precursor to LDL. It therefore follows that inhibitors of MTP would inhibit the synthesis of VLDL and LDL, thereby lowering VLDL levels, LDL levels, cholesterol levels, and triglyceride levels in animals and man.
Canadian Patent Application No. 2,091,102 published Mar. 2, 1994 (corresponding to U.S. application Ser. No. 117,362, filed Sep. 3, 1993 (file DC21b)) which is incorporated herein by reference), reports MTP inhibitors which also block apoB containing lipoprotein secretion in a human hepatic cell line (HepG2 cells). This provides further support for the proposal that an MTP inhibitor would lower apoB containing lipoprotein and lipid levels in vivo. This Canadian patent application discloses a method for identifying the MTP inhibitors.
The use of microsomal triglyceride transfer protein (MTP) inhibitors for decreasing serum lipids including cholesterol and triglycerides and their use in treating atherosclerosis, obesity, hyperglycemia, and pancreatitis is disclosed in WO 96/26205, published Aug. 29, 1996, U.S. application Ser. No. 472,067, filed Jun. 6, 1995 (file DC21e), U.S. application Ser. No. 548,811, filed Jan. 11, 1996 (file DC21h), U.S. provisional application Ser. No. 60/017,224, filed May 9, 1996 (file HX79a*), U.S. provisional application Ser. No. 60/017,253, filed May 10, 1996 (file HX82*), U.S. provisional application Ser. No. 60/017,254, May 10, 1996 (file HX84*) and U.S. provisional application Ser. No. 60/028,216, filed Oct. 1, 1996 (file HX86*).
All of the above U.S. applications are incorporated herein by reference.